Toxoplasmosis, an infection caused by the protozoan parasite Toxoplasma gondii, affects at least one third of the world's human population. There only exists one genus and one species of the parasite Toxoplasma but the parasitic isolates are currently classified into 12 haplogroups I to XII. The strains mostly encountered in Europe and North America are of Type II. Most often the parasite infects warm-blooded animals, including humans, but its definitive host is a felid.
The prevalence of toxoplasmosis varies from one country to another; it is between 60% and 80% in some regions of South America. In Western Europe the prevalence of toxoplasmosis varies from 50 to 70%; it varies from 20 to 50% in Southern Europe and in wet regions in Africa; it is less than 30% in the Scandinavian countries and in the United Kingdom, 25 to 30% in Central European countries and very low in South-East Asia and North America (Pappas et al., 2009).
In France, it is estimated that 40 to 50% of the adult population is infected (source: French National Authority for Health—Haute Autorité de Santé, 2009; Centre National de Reference de la Toxoplasmose (CNR)), generally without any apparent symptoms, and that there are between 200 000 and 300 000 new infections each year, including 27 000 cases in pregnant women. Three hundred foetuses suffering from congenital toxoplasmosis (CNR 2011), of whom 30 will develop serious sequelae, are identified every year in France.
In the USA, the prevalence is reported to be 11% in pregnant women born in the USA and 28.1% for those born outside the border limits (Jones et al., 2007). In this country, 1 child out of 10 000 suffers from congenital toxoplasmosis (Brown et al., 2009; Lopez et al., 2000) and ocular toxoplasmosis is the cause of uveitis in 17% of cases and of posterior uveitis in 25% of cases.
While the infection of immunocompetent individuals with Type II strains is generally benign, it can prove to be dramatic in immunodeficient persons or when the immunity system is not yet functional (congenital disorders). In children suffering from congenital toxoplasmosis the disorders are chiefly cerebral and ocular (hydrocephaly, intra-cranial calcifications, convulsions, mental deficiency, chorioretinitis). In addition, the infection of individuals even if they are immunocompetent can be fatal. Finally, temporal cerebral inflammation which occurs during infection with T. gondii has recently been positively correlated with a group of neuropsychiatric disorders including schizophrenia (Yolken et al., 2009; Pedersen et al., 2011), bi-polar disorders, neuro-degenerative diseases such as Alzheimer's disease (Kusbeci et al., 2011) and Parkinson's disease (Miman et al., 2010).
It is therefore of importance to be able to make early diagnosis of infection with T. gondii so as to set up adequate treatment: combination of pyrimethamine-sulfadiazine and folic acid in patients suffering from congenital toxoplasmosis or in immunodepressed patients, and spiramycin in pregnant women.
The current diagnosis of toxoplasmosis is essentially based on the detection, in the serum of patients, of specific antibodies of G and M isotypes directed against the whole parasite. The level of M immunoglobulins (IgM), in theory the markers of a recent infection, and then of serum G immunoglobulins (IgG) become raised within two weeks following after contamination. While IgMs are the sign of a recent infection (they appear within few days, the peak being reached in 2-3 months after which they decrease), they may persist for several months, even several years and are not therefore reliable indicators of recent seroconversion. In addition, since each patient is different there is no IgG “threshold” level which can be used to distinguish between longstanding infection and recent infection.
The large majority of diagnoses are performed in pregnant women, as part of a multiple diagnosis known as TORCH which targets pathogenic agents able to pass through the placental barrier (T—Toxoplasma gondii, O—Other infections (Coxsackievirus, Syphilis, Varicella-Zoster Virus, HIV, Parvovirus B19), R—Rubella, C—Cytomegalovirus, H—Herpes simplex virus). Positive serological tests are supplemented with an IgG avidity test and the search for the presence of the parasite by polymerase chain reaction (PCR) in amniotic fluid in pregnant women or in cerebrospinal fluid in children (only in some countries but not in France) or in immunodepressed individuals suspected of recent infection (Montoya and Remington, 2008).
Most diagnosis kits marketed for toxoplasmosis use parasitic lysates (whole antigens) for the detection of specific antibodies. These kits are sensitive, specific and can be automated but are subject to variations in quality over time and remain costly on account of the preparation mode of the antigens which are subjected to amplification of the parasites in the intra-peritoneal cavity of mice or in cultured human cells. The sensitivity of the assays is also variable.
The use of recombinant proteins as antigens to sensitize supports used for the detection of specific antibodies is therefore undergoing increasing development. Several antigens of Toxoplasma gondii have been identified and classified into different families in relation to their cell location:                The antigens of toxoplasma dense granules (GRA1, GRA2, GRA3, GRA4, GRA5, GRA6, GRA7, GRA8 . . . )        The surface proteins (SAG1, SAG2, . . . )        The antigens of rhoptria (secretory organelles particular to Apicomplexa) such as ROP1 . . . .        
A first type of test, Architect, based on the detection of the major surface protein of the parasite (SAG1) and the dense granule protein GRA8, is marketed by Abbott. Patent application EP 1 082 343 by Abbott describes a diagnostic composition comprising the antigens p29 (GRA7), p30 (SAG1) and p35 (GRA8), or p29 (GRA7), p35 (GRA8) and p66 (ROP1).
The GRA2/p28 antigens (Mercier et al., 1993; FR 2 692 282) and GRA6/p32 (Lecordier et al., 1993; FR 2 702 497) are antigens derived from dense granules, secreted by T. gondii and playing an essential role in intracellular parasitism. These are major components of the dense granules (secretory organelles particular to Apicomplexa) and of the vacuole in which the parasite multiples, within the infected cell. These highly immunogenic proteins are good candidates for diagnostic applications.
The recombinant protein GRA2, purified from a bacterial production system, has been tested by ELISA assay, the specificity being 96.4% and sensitivity 95.8% to 100% for acute infections, lower for chronic infections (Golkar et al., 2007). The recombinant protein GRA6 has been tested by ELISA assay and shows much better specificity for sera from patients suffering from acute infection than from those suffering from chronic infection (Golkar et al., 2008).
It has been proposed to associate several recombinant antigens to increase test specificity and sensitivity. In particular, the recombinant protein GRA2 has been associated with the ROP1 (P66) antigen; these two antigens are detected more frequently in sera from patients suffering from acute infection than in sera from patients suffering from chronic infection, thereby allowing a distinction to be made between the two clinical cases (Holec-Gasior et al., 2009). The recombinant protein GRA6 has been associated with GRA1, another antigen of dense granules, in an ELISA assay. This assay, intended to differentiate between patients showing a profile of recent infection and those having a chronic infection, has proved to be insufficiently sensitive for this application (Ferrandiz et al., 2004). The combination of the recombinant antigens GRA2 and GRA6 has been used in a vaccine combination but the antigenic stimulation induced by the combination proved to be disappointing compared with that obtained by injection of the GRA2 protein alone (Golkar et al., 2007).
One of the great challenges in the treatment of toxoplasmosis, in particular in pregnant women, is to initiate treatment as early as possible after the primary infection so as to limit as far as possible the risk of the parasite passing through the placental barrier. This is only possible if diagnosis is made very early. Therefore the development of new assays allowing early diagnosis of the infection will meet a true need of the medical profession. Also, the specificity of known assays must be improved since they still detect too many “false-positives”. In particular, for detection in new-borns, it is necessary to distinguish as early as possible between a positive reaction simply due to the presence of maternal antibodies acquired during labour and a true positive reaction related to congenital toxoplasmosis.